The long-term goals of this research are to understand the structure, assembly, and function of cilia and flagella. The studies will utilize Chlamydomonas and mice as model organisms, and will focus on processes and proteins that are highly conserved among ciliated organisms. A combination of genetic, biochemical, and cell biological approaches will be taken. Intraflagellar transport (IFT), which is necessary for assembly of cilia, will be investigated. IFT involves the movement of particles and cargo within the cilium. Studies are proposed to learn more about the motors that transport the IFT particles, the particles themselves, and the BBSome ~ an IFT cargo adapter. Experiments will use existing Chlamydomonas mutants to determine the specific function of the IFT-particle protein IFT74, and of FAP133 ~ the intermediate chain for the retrograde IFT motor, dynein 1B. Studies will Investigate the specific roles of several BBSome proteins and establish the basis for BBSome-IFT particle interaction. The hypotiiesis that the BBSome exports proteins from the flagella will be tested. The relationship between the BBSome and phosphollpase D, a putative cargo of the BBSome, will be investigated. Studies will explore how the transition zone protein CEP290 regulates entry of IFT particles and BBSomes into the flagellum. Studies will define the domains of CEP290 necessary for assembly into the transition zone, assign specific functions to the domains, and Identify interacting proteins. An existing mutant defective In an uncharacterized motility regulating protein will be studied to determine the location of the protein In the axoneme and to Identify Its interacting partners. To discover novel proteins involved in flagellar assembly, in flagellar motility, and In the induction of genes encoding flagellar proteins, new insertlonal mutants will be made by a method that allows rapid identification of the genomic DNA flanking both ends of the insert, so that the mutated gene is quickly identified. Characterization of selected mutants will be facilitated by rescuing the mutants with constructs expressing HA- or GFP-tagged proteins so that the proteins can be quickly localized, observed by TIRF microscopy in living flagella, and followed in biochemical experiments.